Nucleotide-binding kinetics of Na,K-ATPase: Cation dependence

Correlation between the Na,K-ATPase affinity to ADP and the cation (its nature and concentration) present in the medium was investigated. In buffer with low ionic strength (I approximate to 1 mM) high-affinity ADP binding was not observed, while a stepwise increase in the concentrations of added cation (Na+, Tris(+), imidazole(+), N-methylglucamine(+), choline(+)) induced an increase in the ADP affinity. The effect was fully saturated at 30-50 mM for all of the cations tested. The maximal affinity for ADP was slightly higher in the presence of Na+, Tris(+), or imidazole(+) than in the presence of N-methylglucamine(+) or choline(+) (equilibrium dissociation constant K-d 0.2-0.3 vs 0.7 muM). The ADP dissociation rates from its complex with enzyme in the presence of Na+ or Tris(+) were similar, implying identity of the nucleotide-binding enzyme conformations, which therefore are assigned to E-1. The ability to compete with K+ clearly distinguished Na+ from other cations, which speaks against the sole involvement of the transport sites in the induction of the ADP-binding E-1 conformation. Since the cations are similar in their mode of induction of the high ADP affinity but they demonstrate a pronounced difference in ability to compete with K+, their effects cannot be combined within any scheme with only one type of cation-binding sites. We suggest that the high affinity toward nucleotide is induced by cation interactions within the protein or lipid and that these nucleotide-domain-related sites coexist with the transport sites, which bind only Na+ or K+.